Synthesis, deprotection, analysis &amp; purification of RNA &amp; ribozymes

ABSTRACT

Method for purification and synthesis of RNA molecules and enzymatic RNA molecules in enzymatically active form.

[0001] This application is a continuation-in-part of two applications byUsman et al., both entitled “Synthesis, deprotection, analysis andpurification of RNA and ribozymes” and filed on Nov. 28, 1994 as U.S.Ser. No. of 08/345,516, and on May 18, 1994, as U.S. Ser. No.08/245,736, which is a continuation-in-part of Dudycz et al. entitled“Preparation of purified ribozymes in sodium, potassium or magnesiumsalt form”, filed Dec. 14, 1993, U.S. Ser. No. 08/167,586 (pending),which is a continuation of Dudycz et al. entitled “Preparation ofpurified ribozymes in sodium, potassium or magnesium salt form”, filedMay 14, 1992, U.S. Ser. No. 07/884,436 (abandoned). All of these priorapplications are hereby incorporated by reference herein (includingdrawings).

BACKGROUND OF THE INVENTION

[0002] This invention relates to the synthesis, deprotection, andpurification of enzymatic RNA or modified enzymatic RNA molecules inmilligram to kilogram quantities with high biological activity.

[0003] The following is a brief history of the discovery and activity ofenzymatic RNA molecules or ribozymes. This history is not meant to becomplete but is provided only for understanding of the invention thatfollows. This summary is not an admission that all of the work describedbelow is prior art to the claimed invention.

[0004] Prior to the 1970s it was thought that all genes were directlinear representations of the proteins that they encoded. Thissimplistic view implied that all genes were like ticker tape messages,with each triplet of DNA “letters” representing one protein “word” inthe translation. Protein synthesis occurred by first transcribing a genefrom DNA into RNA (letter for letter) and then translating the RNA intoprotein (three letters at a time). In the mid 1970s it was discoveredthat some genes were not exact, linear representations of the proteinsthat they encode. These genes were found to contain interruptions in thecoding sequence which were removed from, or “spliced out” of, the RNAbefore it became translated into protein. These interruptions in thecoding sequence were given the name of intervening sequences (orintrons) and the process of removing them from the RNA was termedsplicing. After the discovery of introns, two questions immediatelyarose: i) why are introns present in genes in the first place, and ii)how do they get removed from the RNA prior to protein synthesis? Thefirst question is still being debated, with no clear answer yetavailable. The second question, how introns get removed from the RNA, ismuch better understood after a decade and a half of intense research onthis question. At least three different mechanisms have been discoveredfor removing introns from RNA. Two of these splicing mechanisms involvethe binding of multiple protein factors which then act to correctly cutand join the RNA. A third mechanism involves cutting and joining of theRNA by the intron itself, in what was the first discovery of catalyticRNA molecules.

[0005] Cech and colleagues were trying to understand how RNA splicingwas accomplished in a single-celled pond organism called Tetrahymenathermophila. They had chosen Tetrahymena thermophila as a matter ofconvenience, since each individual cell contains over 10,000 copies ofone intron-containing gene (the gene for ribosomal RNA). They reasonedthat such a large number of intron-containing RNA molecules wouldrequire a large amount of (protein) splicing factors to get the intronsremoved quickly. Their goal was to purify these hypothesized splicingfactors and to demonstrate that the purified factors could splice theintron-containing RNA in vitro. Cech rapidly succeeded in getting RNAsplicing to work in vitro, but something unusual was going on. Asexpected, splicing occurred when the intron-containing RNA was mixedwith protein-containing extracts from Tetrahymena, but splicing alsooccurred when the protein extracts were left out. Cech proved that theintervening sequence RNA was acting as its own splicing factor to snipitself out of the surrounding RNA. They published this startlingdiscovery in 1982. Continuing studies in the early 1980's served toelucidate the complicated structure of the Tetrahymena intron and todecipher the mechanism by which self-splicing occurs. Many researchgroups helped to demonstrate that the specific folding of theTetrahymena intron is critical for bringing together the parts of theRNA that will be cut and spliced. Even after splicing is complete, thereleased intron maintains its catalytic structure. As a consequence, thereleased intron is capable of carrying out additional cleavage andsplicing reactions on itself (to form intron circles). By 1986, Cech wasable to show that a shortened form of the Tetrahymena intron could carryout a variety of cutting and joining reactions on other pieces of RNA.The demonstration proved that the Tetrahymena intron can act as a trueenzyme: i) each intron molecule wa§able to cut many substrate moleculeswhile the intron molecule remained unchanged, and ii) reactions werespecific for RNA molecules that contained a unique sequence (CUCU) whichallowed the intron to recognize and bind the RNA. Zaug and Cech coinedthe term “ribozyme” to describe any ribonucleic acid molecule that hasenzyme-like properties. Also in 1986, Cech showed that the RNA substratesequence recognized by the Tetrahymena ribozyme could be changed byaltering a sequence within the ribozyme itself. This property has led tothe development of a number of site-specific ribozymes that have beenindividually designed to cleave at other RNA sequences. The Tetrahymenaintron is the most well-studied of what is now recognized as a largeclass of introns, Group I introns. The overall folded structure,including several sequence elements, is conserved among the Group Iintrons, as is the general mechanism of splicing. Like the Tetrahymenaintron, some members of this class are catalytic, i e. the intron itselfis capable of the self-splicing reaction. Other Group I introns requireadditional (protein) factors, presumably to help the intron fold intoand/or maintain its active structure. While the Tetrahymena intron isrelatively large, (413 nucleotides) a shortened form of at least oneother catalytic intron (SunY intron of phage T4, 180 nucleotides) mayprove advantageous not only because of its smaller size but because itundergoes self-splicing at an even faster rate than the Tetrahymenaintron.

[0006] Ribonuclease P (RNAseP) is an enzyme comprised of both RNA andprotein components which are responsible for converting precursor tRNAmolecules into their final form by trimming extra RNA off one of theirends. RNAseP activity has been found in all organisms tested, but thebacterial enzymes have been the most studied. The function of RNAseP hasbeen studied since the mid-1970s by many labs. In the late 1970s, SidneyAltman and his colleagues showed that the RNA component of RNAseP isessential for its processing activity; however, they also showed thatthe protein component also was required for processing under theirexperimental conditions. After Cech's discovery of self-splicing by theTetrahymena intron, the requirement for both protein and RNA componentsin RNAseP was reexamined. In 1983, Altman and Pace showed that the RNAwas the enzymatic component of the RNAseP complex. This demonstratedthat an RNA molecule was capable of acting as a true enzyme, processingnumerous tRNA molecules without itself undergoing any change. The foldedstructure of RNAseP RNA has been determined, and while the sequence isnot strictly conserved between RNAs from different organisms, thishigher order structure is. It is thought that the protein component ofthe RNAseP complex may serve to stabilize the folded RNA in vivo Atleast one RNA position important both to substrate recognition and todetermination of the cleavage site has been identified, however littleelse is known about the active site. Because tRNA sequence recognitionis minimal, it is clear that some aspect(s) of the tRNA structure mustalso be involved in substrate recognition and cleavage activity. Thesize of RNAseP RNA (>350 nucleotides), and the complexity of thesubstrate recognition, may limit the potential for the use of anRNAseP-like RNA in therapeutics. However, the size of RNAseP is beingtrimmed down (a molecule of only 290 nucleotides functions reasonablywell). In addition, substrate recognition has been simplified by therecent discovery that RNAseP RNA can cleave small RNAs lacking thenatural tRNA secondary structure if an additional RNA (containing a“guide” sequence and a sequence element naturally present at the end ofall tRNAs) is present as well.

[0007] Symons and colleagues identified two examples of a self-cleavingRNA that differed from other forms of catalytic RNA already reported.Symons was studying the propagation of the avocado sunblotch viroid(ASV), an RNA virus that infects avocado plants. Symons demonstratedthat as little as 55 nucleotides of the ASV RNA was capable of foldingin such a way as to cut itself into two pieces. It is thought that invivo self-cleavage of these RNAs is responsible for cutting the RNA intosingle genome-length pieces during viral propagation. Symons discoveredthat variations on the minimal catalytic sequence from ASV could befound in a number of other plant pathogenic RNAs as well. Comparison ofthese sequences revealed a common structural design consisting of threestems and loops connected by central loop containing many conserved(invariant from one RNA to the next) nucleotides. The predictedsecondary structure for this catalytic RNA reminded the researchers ofthe head of a hammer consisting of three double helical domains, stemsI, II and III and a catalytic core (FIGS. 1 and 2a); thus it was namedas such. Uhlenbeck was successful in separating the catalytic region ofthe ribozyme from that of the substrate. Thus, it became possible toassemble a hammerhead ribozyme from 2 (or 3) small synthetic RNAs. A19-nucleotide catalytic region and a 24-nucleotide substrate,representing division of the hammerhead domain along the axes of stems Iand II (FIG. 2b) were sufficient to support specific cleavage. Thecatalytic domain of numerous hammerhead ribozymes have now been studiedby both the Uhlenbeck and Symons groups with regard to defining thenucleotides required for specific assembly and catalytic activity anddetermining the rates of cleavage under various conditions.

[0008] Haseloff and Gerlach showed it was possible to divide the domainsof the hammerhead ribozyme in a different manner, division of thehammerhead domain along the axes of stems I and III (FIG. 2c). By doingso, they placed most of the required sequences in the strand that didn'tget cut (the ribozyme) and only a required UH where H═C, A, U in thestrand that did get cut (the substrate). This resulted in a catalyticribozyme that could be designed to cleave any UH RNA sequence embeddedwithin a longer “substrate recognition” sequence. The specific cleavageof a long mRNA, in a predictable manner using several such hammerheadribozymes, was reported in 1988. A further development was the divisionof the catalytic hammerhead domain along the axes of stems III and II(FIG. 2d, Jeffries and Symons, Nucleic Acids. Res. 1989, 17, 1371-1377.)

[0009] One plant pathogen RNA (from the negative strand of the tobaccoringspot virus) undergoes self-cleavage but cannot be folded into theconsensus hammerhead structure described above. Bruening and colleagueshave independently identified a 50-nucleotide catalytic domain for thisRNA. In 1990, Hampel and Tritz succeeded in dividing the catalyticdomain into two parts that could act as substrate and ribozyme in amultiple-turnover, cutting reaction (FIG. 3). As with the hammerheadribozyme, the hairpin catalytic portion contains most of the sequencesrequired for catalytic activity while only a short sequence (GUC in thiscase) is required in the target. Hampel and Tritz described the foldedstructure of this RNA as consisting of a single hairpin and coined theterm “hairpin” ribozyme (Bruening and colleagues use the term“paperclip” for this ribozyme motif, see, FIG. 3). Continuingexperiments suggest an increasing number of similarities between thehairpin and hammerhead ribozymes in respect to both binding of targetRNA and mechanism of cleavage. At the same time, the minimal size of thehairpin ribozyme is still 50-60% larger than the minimal hammerheadribozyme.

[0010] Hepatitis Delta Virus (HDV) is a virus whose genome consists ofsingle-stranded RNA. A small region (˜80 nucleotides, FIG. 4) in boththe genomic RNA, and in the complementary anti-genomic RNA, issufficient to support self-cleavage. As the most recently discoveredribozyme, HDV's ability to self-cleave has only been studied for a fewyears, but is interesting because of its connection to a human disease.In 1991, Been and Perrotta proposed a secondary structure for the HDVRNAs that is conserved between the genomic and anti-genomic RNAs and isnecessary for catalytic activity. Separation of the HDV RNA into“ribozyme” and “substrate” portions has recently been achieved by Been,but the rules for targeting different substrate RNAs have not yet beendetermined fully (see, FIG. 4). Been has also succeeded in reducing thesize of the HDV ribozyme to −60 nucleotides.

[0011] The Table I lists some of the characteristics of the ribozymesdiscussed above.

[0012] Ribozymes are RNA molecules having an enzymatic activity which isable to repeatedly cleave other separate RNA molecules in a nucleotidebase sequence specific manner. It is said that such enzymatic RNAmolecules can be targeted to virtually any RNA transcript and efficientcleavage has been achieved in vitro. Kim et al., 84 Proc. Nat. Acad. ofSci. USA 8788, 1987; Haseloff and Gerlach, 334 Nature 585, 1988; Cech,260 JAMA 3030, 1988; and Jefferies et al., 17 Nucleic Acid Research1371, 1989.

[0013] Six basic varieties of naturally-occurring enzymatic RNAs areknown presently. Each can catalyze the hydrolysis of RNA phosphodiesterbonds in trans (and thus can cleave other RNA molecules) underphysiological conditions. Table I summarizes some of the characteristicsof these ribozymes. In general, enzymatic nucleic acids act by firstbinding to a target RNA. Such binding occurs through the target bindingportion of a enzymatic nucleic acid which is held in close proximity toan enzymatic portion of the molecule that acts to cleave the target RNA.Thus, the enzymatic nucleic acid first recognizes and then binds atarget RNA through complementary base-pairing, and once bound to thecorrect site, acts enzymatically to cut the target RNA. Strategiccleavage of such a target RNA will destroy its ability to directsynthesis of an encoded protein. After an enzymatic nucleic acid hasbound and cleaved its RNA target, it is released from that RNA to searchfor another target and can repeatedly bind and cleave new targets.

[0014] By “enzymatic RNA molecule” it is meant an RNA molecule which hascomplementarity in a substrate binding region to a specified mRNAtarget, and also has an enzymatic activity which is active tospecifically cleave that mRNA. That is, the enzymatic RNA molecule isable to intermolecularly cleave mRNA and thereby inactivate a targetmRNA molecule. This complementarity functions to allow sufficienthybridization of the enzymatic RNA molecule to the target RNA to allowthe cleavage to occur. One hundred percent complementarity is preferred,but complementarity as low as 50-75% may also be useful in thisinvention. For in vivo treatment, complementarity between 30 and 45bases is preferred; although lower numbers are also useful.

[0015] By “complementary” is meant a nucleotide sequence that can formhydrogen bond(s) with other nucleotide sequence by either traditionalWatson-Crick or other non-traditional types (for example Hoogsteen type)of base-paired interactions.

[0016] The enzymatic nature of a ribozyme is advantageous over othertechnologies, such as antisense technology (where a nucleic acidmolecule simply binds to a nucleic acid target to block its translation)since the concentration of ribozyme necessary to affect a therapeutictreatment is lower than that of an antisense oligonucleotide. Thisadvantage reflects the ability of the ribozyme to act enzymatically.Thus, a single ribozyme molecule is able to cleave many molecules oftarget RNA. In addition, the ribozyme is a highly specific inhibitor,with the specificity of inhibition depending not only on the basepairing mechanism of binding to the target RNA, but also on themechanism of target RNA cleavage. Single mismatches, orbase-substitutions, near the site of cleavage can completely eliminatecatalytic activity of a ribozyme. Similar mismatches in antisensemolecules do not prevent their action (Woolf, T. M., et al., 1992, Proc.Natl. Acad. Sci. USA, 89, 7305-7309). Thus, the specificity of action ofa ribozyme is greater than that of an antisense oligonucleotide bindingthe same RNA site.

[0017] In preferred embodiments of this invention, the enzymatic nucleicacid molecule is formed in a hammerhead or hairpin motif, but may alsobe formed in the motif of a hepatitis delta virus, group I intron orRNaseP RNA (in association with an RNA guide sequence) or Neurospora VSRNA. Examples of such hammerhead motifs are described by Rossi et al.,1992, Aids Research and Human Retroviruses 8, 183, of hairpin motifs byHampel et al., EPA 0360257, Hampel and Tritz, 1989 Biochemistry 28,4929, and Hampel et al., 1990 Nucleic Acids Res. 18, 299, and an exampleof the hepatitis delta virus motif is described by Perrotta and Been,1992 Biochemistry 31, 16; of the RNaseP motif by Guerrier-Takada et al.,1983 Cell 35, 849, Neurospora VS RNA ribozyme motif is described byCollins (Saville and Collins, 1990 Cell 61, 685-696; Saville andCollins, 1991 Proc. Natl. Acad. Sci, USA 88, 8826-8830; Collins andOlive, 1993 Biochemistry 32, 2795-2799) and of the Group I intron byCech et al., U.S. Pat. No. 4,987,071. These specific motifs are notlimiting in the invention and those skilled in the art will recognizethat all that is important in an enzymatic nucleic acid molecule of thisinvention which is complementary to one or more of the target gene RNAregions, and that it have nucleotide sequences within or surroundingthat substrate binding site which impart an RNA cleaving activity to themolecule.

[0018] Enzymatic nucleic acids act by first binding to a target RNA (orDNA, see Cech U.S. Pat. No. 5,180,818). Such binding occurs through thetarget binding portion of an enzymatic nucleic acid which is held inclose proximity to an enzymatic portion of molecule that acts to cleavethe target RNA. Thus, the enzymatic nucleic acid first recognizes andthen binds a target RNA through complementary base-pairing, and oncebound to the correct site, acts enzymatically to cut the target RNA.Cleavage of such a target RNA will destroy its ability to directsynthesis of an encoded protein. After an enzymatic nucleic acid hasbound and cleaved its RNA target it is released from that RNA to searchfor another target and can repeatedly bind and cleave new targets.

[0019] The invention provides a method for producing a class ofenzymatic cleaving agents or antisense molecules which exhibit a highdegree of specificity for the RNA or DNA of a desired target. Theenzymatic nucleic acid or antisense molecule is preferably targeted to ahighly conserved sequence region of a target such that specifictreatment of a disease or condition can be provided with a singleenzymatic nucleic acid. Such, nucleic acid molecules can be deliveredexogenously to specific cells as required. In the preferred hammerheadmotif the small size (less than 60 nucleotides, preferably between 30-40nucleotides in length) of the molecule allows the cost of treatment tobe reduced compared to other ribozyme motifs.

[0020] Synthesis of nucleic acids greater than 100 nucleotides in lengthis difficult using automated methods, and the therapeutic cost of suchmolecules is prohibitive. In this invention, small enzymatic nucleicacid motifs (e.g., of the hammerhead structure) are used for exogenousdelivery.

[0021] The simple structure of these molecules increases the ability ofthe enzymatic nucleic acid to invade targeted regions of the mRNAstructure. Unlike the situation when the hammerhead structure isincluded within longer transcripts, there are no non-enzymatic nucleicacid flanking sequences to interfere with correct folding of theenzymatic nucleic acid structure or with complementary regions.

[0022] Generally, RNA is synthesized and purified by methodologies basedon: tetrazole to activate the RNA amidite, NH₄OH to remove the exocyclicamino protecting groups, tetra-n-butylammonium fluoride (TBAF) to removethe 2′-OH alkylsilyl protecting groups, and gel purification andanalysis of the deprotected RNA. In particular this applies to, but isnot limited to, a certain class of RNA molecules, ribozymes. These maybe formed either chemically or using enzymatic methods. Examples of thechemical synthesis, deprotection, purification and analysis proceduresare provided by Usman et al., 1987 J. American Chem. Soc., 109, 7845,Scaringe et al. Nucleic Acids Res. 1990, 18, 5433-5341, Perreault et al.Biochemistry 1991, 30 4020-4025, and Slim and Gait Nucleic Acids Res.1991, 19, 1183-1188. Odai et al. FEBS Lett. 1990, 267, 150-152 describesa reverse phase chromatographic purification of RNA fragments used toform a ribozyme. All the above noted references are all herebyincorporated by reference herein.

[0023] The aforementioned chemical synthesis, deprotection, purificationand analysis procedures are time consuming (10-15 m coupling times) andmay also be affected by inefficient activation of the RNA amidites bytetrazole, time consuming (6-24 h) and incomplete deprotection of theexocyclic amino protecting groups by NH₄OH, time consuming (6-24 h),incomplete and difficult to desalt TBAF-catalyzed removal of thealkylsilyl protecting groups, time consuming and low capacitypurification of the RNA by gel electrophoresis, and low resolutionanalysis of the RNA by gel electrophoresis.

[0024] Imazawa and Eckstein, 1979 J. Org. Chem., 12, 2039, describe thesynthesis of 2′-amino-2′-deoxyribofuranosyl purines. They state that

[0025] “To protect the 2′-amino function, we selected thetrifluoroacetyl group which can easily be removed.”

SUMMARY OF THE INVENTION

[0026] This invention concerns -the chemical synthesis, deprotection,and purification of RNA, enzymatic RNA or modified RNA molecules ingreater than milligram quantities with high biological activity.Applicant has determined that the synthesis of enzymatically active RNAin high yield and quantity is dependent upon certain critical steps usedduring its preparation. Specifically, it is important that the RNAphosphoramidites are coupled efficiently in terms of both yield andtime, that correct exocyclic amino protecting groups be used, that theappropriate conditions for the removal of the exocyclic amino protectinggroups and the alkylsilyl protecting groups on the 2′-hydroxyl are used,and that the correct work-up and purification procedure of the resultingribozyme be used.

[0027] To obtain a correct synthesis in terms of yield and biologicalactivity of a large RNA molecule (i.e., about 30 to 40 nucleotidebases), the protection of the amino functions of the bases requireseither amide or substituted amide protecting groups, which must be, onthe one hand, stable enough to survive the conditions of synthesis, andon the other hand, removable at the end of the synthesis. Theserequirements are met by the amide protecting groups shown in FIG. 6, inparticular, benzoyl for adenosine, isobutyryl or benzoyl for cytidine,and isobutyryl for guanosine, which may be removed at the end of thesynthesis by incubating the RNA in NH₃/EtOH (ethanolic ammonia) for 20 hat 65° C. In the case of the phenoxyacetyl type protecting groups shownin FIG. 6 on guanosine and adenosine and acetyl protecting groups oncytidine, an incubation in ethanolic ammonia for 4 h at 65° C. is usedto obtain complete removal of these protecting groups. Removal of thealkylsilyl 2′-hydroxyl protecting groups can be accomplished using atetrahydrofuran solution of TBAF at room temperature for 8-24 h.

[0028] The most quantitative procedure for recovering the fullydeprotected RNA molecule is by either ethanol precipitation, or an anionexchange cartridge desalting, as described in Scaringe et al. NucleicAcids Res. 1990, 18, 5433-5341. The purification of the long RNAsequences may be accomplished by a two-step chromatographic procedure inwhich the molecule is first purified on a reverse phase column witheither the trityl group at the 5′ position on or off. This purificationis accomplished using an acetonitrile gradient with triethylammonium orbicarbonate salts as the aqueous phase. In the case of the trityl onpurification, the trityl group may be removed by the addition of an acidand drying of the partially purified RNA molecule. The finalpurification is carried out on an anion exchange column, using alkalimetal perchlorate salt gradients to elute the fully purified RNAmolecule as the appropriate metal salts, e.g. Na⁺, Li⁺ etc. A finalde-salting step on a small reverse-phase cartridge completes thepurification procedure. Applicant has found that such a procedure notonly fails to adversely affect activity of a ribozyme, but may improveits activity to cleave target RNA molecules.

[0029] Applicant has also determined that significant (see Tables 2-4)improvements in the yield of desired full length product (FLP) can beobtained by:

[0030] 1. Using 5-S-alkyltetrazole at a delivered or effectiveconcentration of 0.25-0.5 M or 0.15-0.35 M for the activation of the RNA(or analogue) amidite during the coupling step. (By delivered is meantthat the actual amount of chemical in the reaction mix is known. This ispossible for large scale synthesis since the reaction vessel is of sizesufficient to allow such manipulations. The term effective means thatavailable amount of chemical actually provided to the reaction mixturethat is able to react with the other reagents present in the mixture.Those skilled in the art will recognize the meaning of these terms fromthe examples provided herein.) The time for this step is shortened from10-15 m, vide supra, to 5-10 m. Alkyl, as used herein, refers to asaturated aliphatic hydrocarbon, including straight-chain,branched-chain, and cyclic alkyl groups. Preferably, the alkyl group has1 to 12 carbons. More preferably it is a lower alkyl of from 1 to 7carbons, more preferably 1 to 4 carbons. The alkyl group may besubstituted or unsubstituted. When substituted the substituted group(s)is, preferably, hydroxyl, cyano, alkoxy, ═O, ═S, NO₂ or N(CH₃)₂, amino,or SH. The term also includes alkenyl groups which are unsaturatedhydrocarbon groups containing at least one carbon-carbon double bond,including straight-chain, branched-chain, and cyclic groups. Preferably,the alkenyl group has 1 to 12 carbons. More preferably it is a loweralkenyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. Thealkenyl group may be substituted or unsubstituted. When substituted thesubstituted group(s) is preferably, hydroxyl, cyano, alkoxy, ═O, ═S,NO₂, halogen, N(CH₃)₂, amino, or SH. The term “alkyl” also includesalkynyl groups which have an unsaturated hydrocarbon group containing atleast one carbon-carbon triple bond, including straight-chain,branched-chain, and cyclic groups. Preferably, the alkynyl group has 1to 12 carbons. More preferably it is a lower alkynyl of from 1 to 7carbons, more preferably 1 to 4 carbons. The alkynyl group may besubstituted or unsubstituted. When substituted the substituted group(s)is preferably, hydroxyl, cyano, alkoxy, ═O, ═S, NO₂ or N(CH₃)₂, amino orSH.

[0031] Such alkyl groups may also include aryl, alkylaryl, carbocyclicaryl, heterocyclic aryl, amide and ester groups. An “aryl” group refersto an aromatic group which has at least one ring having a conjugated πelectron system and includes carbocyclic aryl, heterocyclic aryl andbiaryl groups, all of which may be optionally substituted. The preferredsubstituent(s) of aryl groups are halogen, trihalomethyl, hydroxyl, SH,OH, cyano, alkoxy, alkyl, alkenyl, alkynyl, and amino groups. An“alkylaryl” group refers to an alkyl group (as described above)covalently joined to an aryl group (as described above. Carbocyclic arylgroups are groups wherein the ring atoms on the aromatic ring are allcarbon atoms. The carbon atoms are optionally substituted. Heterocyclicaryl groups are groups having from 1 to 3 heteroatoms as ring atoms inthe aromatic ring and the remainder of the ring atoms are carbon atoms.Suitable heteroatoms include oxygen, sulfur, and nitrogen, and includefuranyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolo, pyrimidyl,pyrazinyl, imidazolyl and the like, all optionally substituted. An“amide” refers to an —C(O)—NH—R, where R is either alkyl, aryl,alkylaryl or hydrogen. An “ester” refers to an —C(O)—OR′, where R iseither alkyl, aryl, alkylaryl or hydrogen.

[0032] 2. Using 5-S-alkyltetrazole at an effective, or final,concentration of 0.1-0.35 M for the activation of the RNA (or analogue)amidite during the coupling step. The time for this step is shortenedfrom 10-15 m, vide supra,, to 5-10 m.

[0033] 3. Using alkylamine (MA, where alkyl is preferably methyl, ethyl,propyl or butyl) or NH₄OH/alkylamine (AMA, with the same preferred alkylgroups as noted for MA) @ 65° C. for 10-15 m to remove the exocyclicamino protecting groups (vs 4-20 h @ 55-65° C. using NH₄OH/EtOH orNH₃/EtOH, vide supra). Other alkylamines, e.g. ethylamine, propylamine,butylamine etc. may also be used.

[0034] 4. Using anhydrous triethylamine.hydrogen fluoride (aHF.TEA) @65°C. for 0.5-1.5 h to remove the 2′-hydroxyl alkylsilyl protecting group(vs 8-24 h using TBAF, vide supra or TEA.3HF for 24 h (Gasparutto et al.Nucleic Acids Res. 1992, 20, 5159-5166). Other alkylamine.HF complexesmay also be used, e.g. trimethylamine or diisopropylethylamine.

[0035] 5. The use of anion-exchange resins to purify and/or analyze thefully deprotected RNA. These resins include, but are not limited to,quartenary or tertiary amino derivatized stationary phases such assilica or polystyrene. Specific examples include Dionex-NA100®, Mono-Q®,Poros-Q®.

[0036] Thus, in various aspects, the invention features an improvedmethod for the coupling of RNA phosphoramidites; for the removal ofamide or substituted amide protecting groups; and for the removal of2′-hydroxyl alkylsilyl protecting groups. Such methods enhance theproduction of RNA or analogs of the type described above (e.g., withsubstituted 2′-groups), and allow efficient synthesis of large amountsof such RNA. Such RNA may also have enzymatic activity and be purifiedwithout loss of that activity. While specific examples are given herein,those in the art will recognize that equivalent chemical reactions canbe performed with the alternative chemicals noted above, which can beoptimized and selected by routine experimentation.

[0037] In another aspect, the invention features an improved method forthe purification or analysis of RNA or enzymatic RNA molecules (e.g.28-70 nucleotides in length) by passing said RNA or enzymatic RNAmolecule over an HPLC, e.g., reverse phase and/or an anion exchangechromatography column. The method of purification improves the catalyticactivity of enzymatic RNAs over the gel purification method (see FIG.8).

[0038] This invention also features a method for preparation of pureenzymatically active ribozymes (of size between 28 and 70 nucleotidebases) in sodium, potassium or magnesium salt form by a two steppurification method. Generally the method is applicable to bothsynthetically and enzymatically produced ribozymes, and entails use ofhigh performance liquid chromatography (HPLC) techniques on reversephase columns. Unlike gel purification, HPLC purification as describedin this application can be applied to virtually unlimited amounts ofpurified material. This allows generation of kilogram quantities ofribozymes in each purification batch.

[0039] Thus, in another aspect, the invention features a method forpurification of an enzymatic RNA molecule of 28-70 nucleotide bases bypassing that enzymatic RNA molecule over a high pressure liquidchromatography column. Surprisingly, applicant has determined thatenzymatically active ribozymes can be purified in the desired salt formby the described HPLC or anion exchange methodology.

[0040] In preferred embodiments, the method includes passing theenzymatically active RNA molecule over a reverse phase HPLC column; theenzymatically active RNA molecule is produced in a synthetic chemicalmethod and not by an enzymatic process; and the enzymatic RNA moleculecontains a 5′-DMT group, and the 5′-DMT-containing enzymatically activeRNA molecule is passed over a reverse phase HPLC column to separate itfrom other RNA molecules.

[0041] In a related aspect, the invention features pure ribozyme in aNa⁺, K⁺, or Mg²⁺ salt form. By “pure” is meant that the ribozyme ispreferably provided free of other contaminants, and is at least 85% inthe desired salt form.

[0042] Thus, the purification of long RNA molecules may be accomplishedusing anion exchange chromatography, particularly in conjunction withalkali perchlorate salts. This system may be used to purify very longRNA molecules. In particular, it is advantageous to use a DionexNucleoPak 100© or a Pharmacia Mono Q® anion exchange column for thepurification of RNA by the anion exchange method. This anion exchangepurification may be used following a reverse-phase purification or priorto reverse phase purification. This method results in the formation of asodium salt of the ribozyme during the chromatography. Replacement ofthe sodium alkali earth salt by other metal salts, e.g., lithium,magnesium or calcium perchlorate, yields the corresponding salt of theRNA molecule during the purification.

[0043] In the case of the 2-step purification procedure, in which thefirst step is a reverse phase purification followed by an anion exchangestep, the reverse phase purification is best accomplished usingpolymeric, e.g. polystyrene based, reverse-phase media, using either a5′-trityl-on or 5′-trityl-off method. Either molecule may be recoveredusing this reverse-phase method, and then, once detritylated, the twofractions may be pooled and then submitted to an anion exchangepurification step as described above.

[0044] The method includes passing the enzymatically active RNA moleculeover a reverse phase HPLC column; the enzymatically active RNA moleculeis produced in a synthetic chemical method and not by an enzymaticprocess; and the enzymatic RNA molecule is partially blocked, and thepartially blocked enzymatically active RNA molecule is passed over areverse phase HPLC column to separate it from other RNA molecules.

[0045] In more preferred embodiments, the enzymatically active RNAmolecule, after passage over the reverse phase HPLC column, isdeprotected and passed over a second reverse phase HPLC column (whichmay be the same as the reverse phase HPLC column), to remove theenzymatic RNA molecule from other components. In addition, the column isa silica or organic polymer-based C4, C8 or Cl 8 column having aporosity of at least 125 Å, preferably 300 Å, and a particle size of atleast 2 μm, preferably 5 μm.

[0046] Other features and advantages of the invention will be apparentfrom the following description of the preferred embodiments thereof, andfrom the claims.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0047] The drawings will first briefly be described.

DRAWINGS

[0048]FIG. 1 is a diagrammatic representation of the hammerhead ribozymedomain known in the art. Stem II can be≧2 base-pairs long.

[0049]FIG. 2a is a diagrammatic representation of the hammerheadribozyme domain known in the art; FIG. 2b is a diagrammaticrepresentation of the hammerhead ribozyme as divided by Uhlenbeck (1987,Nature, 327, 596-600) into a substrate and enzyme portion; FIG. 2c is asimilar diagram showing the hammerhead divided by Haseloff and Gerlach(1988, Nature , 334, 585-591) into two portions; and FIG. 2d is asimilar diagram showing the hammerhead divided by Jeffries and Symons(1989, Nucl. Acids. Res., 17, 1371-1371) into two portions.

[0050]FIG. 3 is a diagrammatic representation of the general structureof a hairpin ribozyme. Helix 2 (H2) is provided with a least 4 basepairs (i.e., n is 1, 2, 3 or 4) and helix 5 can be optionally providedof length 2 or more bases (preferably 3-20 bases, i.e., m is from 1-20or more). Helix 2 and helix 5 may be covalently linked by one or morebases (i.e., r is >1 base). Helix 1, 4 or 5 may also be extended by 2 ormore base pairs (e.g., 4-20 base pairs) to stabilize the ribozymestructure, and preferably is a protein binding site. In each instance,each N and N′ independently is any normal or modified base and each dashrepresents a potential base-pairing interaction. These nucleotides maybe modified at the sugar, base or phosphate. Complete base-pairing isnot required in the helices, but is preferred. Helix 1 and 4 can be ofany size (I.e., o and p is each independently from 0 to any number,e.g., 20) as long as some base-pairing is maintained. Essential basesare shown as specific bases in the structure, but those in the art willrecognize that one or more may be modified chemically (a basic, base,sugar and/or phosphate modifications) or replaced with another basewithout significant effect. Helix 4 can be formed from two separatemolecules, i.e., without a connecting loop. The connecting loop whenpresent may be a ribonucleotide with or without modifications to itsbase, sugar or phosphate. “q” is ≧2 bases. The connecting loop can alsobe replaced with a non-nucleotide linker molecule. H refers to bases A,U or C. Y refers to pyrimidine bases. “______” refers to a chemicalbond.

[0051]FIG. 4A is a representation of the general structure of thehepatitis delta virus ribozyme domain known in the art.

[0052]FIG. 4B is a representation of the general structure of theself-cleaving VS RNA ribozyme domain.

[0053]FIG. 5 is a diagrammatic representation of the solid-phasesynthesis of RNA.

[0054]FIG. 6 is a diagrammatic representation of exocyclic aminoprotecting groups for nucleic acid synthesis.

[0055]FIG. 7 is a diagrammatic representation of the deprotection ofRNA.

[0056]FIG. 8 is a graphical representation of the cleavage of an RNAsubstrate by ribozymes synthesized, deprotected and purified using theimproved methods described herein.

[0057]FIGS. 9 and 10 are copies of HPLC results showing purification ofribozyme from failure sequences (FIG. 9) and other contaminants (FIG.10).

[0058]FIG. 11 is a schematic representation of a two pot deprotectionprotocol. Base deprotection is carried out with aqueous methyl amine at65° C. for 10 min. The sample is dried in a speed-vac for 2-24 hoursdepending on the scale of RNA synthesis. Silyl protecting group at the2′-hydroxyl position is removed by treating the sample with 1.4 Manhydrous HF at 65° C. for 1.5 hours.

[0059]FIG. 12 is a schematic representation of a one pot deprotection ofRNA synthesized using RNA phosphoramidite chemistry. Anhydrous methylamine is used to deprotect bases at 65° C. for 15 min. The sample isallowed to cool for 10 min before adding TEA.3HF reagent, to the samepot, to remove protecting groups at the 2′-hydroxyl position. Thedeprotection is carried out for 1.5 hours.

[0060]FIG. 13 is a HPLC profile of a 36 nt long ribozyme, targeted tosite A. The RNA is deprotected using either the two pot or the one potdeprotection protocol. The peaks corresponding to full-length RNA isindicated.

[0061]FIG. 14 is a graph comparing RNA cleavage activity of ribozymesdeprotected by two pot vs one pot deprotection protocols.

[0062]FIG. 15 is a schematic representation of an improved method ofsynthesizing RNA containing phosphorothioate linkages.

[0063]FIG. 16 shows RNA cleavage reaction catalyzed by ribozymescontaining phosphorothioate linkages. Hammerhead ribozyme targeted tosite A is synthesized such that 4 nts at the 5′ end containphosphorothioate linkages. P═O refers to ribozyme withoutphosphorothioate linkages. P═S refers to ribozyme with phosphorothioatelinkages.

[0064]FIG. 17 is a schematic representation of synthesis of2′-N-phtalimido-nucleoside phosphoramidite.

EXAMPLES

[0065] The following are non-limiting examples showing the synthesis ofRNA-containing nucleic acids and the testing of the enzymatic activityof these molecules when they are catalytic RNAs.

[0066] Activation

[0067] The synthesis of RNA molecules may be accomplished chemically orenzymatically. In the case of chemical synthesis the use of tetrazole asan activator of RNA phosphoramidites is known (Usman et al. J. Am. Chem.Soc. 1987, 109, 7845-7854). In this, and subsequent reports, a 0.5 Msolution of tetrazole is allowed to react with the RNA phosphoramiditeand couple with the polymer bound 5′-hydroxyl group for 10 m. Applicanthas determined that using 0.25-0.5 M solutions of 5-S-alkyltetrazolesfor only 5 min gives equivalent or better results. The followingexemplifies the procedure.

Example 1 Synthesis of RNA and Ribozymes Using 5-S-Alkyltetrazoles asActivating Agent

[0068] The method of synthesis used follows the general procedure forRNA synthesis as described in Usman et al., 1987supra and in Scaringe etal., Nucleic Acids Res. 1990, 18, 5433-5441 and makes use of commonnucleic acid protecting and coupling groups, such as dimethoxytrityl atthe 5′-end, and phosphoramidites at the 3′-end. The major differenceused was the activating agent, 5-S-ethyl or -methyltetrazole @0.25 Mconcentration for 5 min.

[0069] All small scale syntheses were conducted on a 394 (ABI)synthesizer using a modified 2.5 μmol scale protocol with a reduced 5min coupling step for alkylsilyl protected RNA and 2.5 m coupling stepfor 2′-O-methylated RNA. A 6.5-fold excess (162.5 μL of 0.1 M=32.5 μmol)of phosphoramidite and a 40-fold excess of S-ethyl tetrazole (400 μL of0.25 M=100 μmol) relative to polymer-bound 5′-hydroxyl was used in eachcoupling cycle. Average coupling yields on the 394, determined bycolorimetric quantitation of the trityl fractions, was 97.5-99%. Otheroligonucleotide synthesis reagents for the 394: Detritylation solutionwas 2% TCA in methylene chloride; capping was performed with 16%N-Methyl imidazole in THF and 10% acetic anhydride/10% 2,6-lutidine inTHF; oxidation solution was 16.9 mM 12, 49 mM pyridine, 9% water in THF.Fisher Synthesis Grade acetonitrile was used directly from the reagentbottle. S-Ethyl tetrazole solution (0.25 M in acetonitrile) was made upfrom the solid obtained from Applied Biosystems.

[0070] All large scale syntheses were conducted on a modified (eightamidite port capacity) 390Z (ABI) synthesizer using a 25 μmol scaleprotocol with a 5-15 min coupling step for alkylsilyl protected RNA and7.5 m coupling step for 2′-O-methylated RNA. A six-fold excess (1.5 mLof 0.1 M=150 μmol) of phosphoramidite and a forty-five-fold excess ofS-ethyl tetrazole (4.5 mL of 0.25 M=1125 μmol) relative to polymer-bound5′-hydroxyl was used in each coupling cycle. Average coupling yields onthe 390Z, determined by calorimetric quantitation of the tritylfractions, was 95.0-96.7%. Oligonucleotide synthesis reagents for the390Z: Detritylation solution was 2% DCA in methylene chloride; cappingwas performed with 16% N-Methyl imidazole in THF and 10% aceticanhydride/10% 2,6-lutidine in THF; oxidation solution was 16.9 mM 12, 49mM pyridine, 9% water in THF. Fisher Synthesis Grade acetonitrile wasused directly from the reagent bottle. S-Ethyl tetrazole solution(0.25-0.5 M in acetonitrile) was made up from the solid obtained fromApplied Biosystems.

[0071] Table 1 is a summary of the results obtained using theimprovements outlined in this application for the large-scale synthesisof RNA and modified RNAs.

[0072] Deprotection

[0073] The first step of the deprotection of RNA molecules may beaccomplished by removal of the exocyclic amino protecting groups witheither NH₄OH/EtOH:3/1 (Usman et al. J. Am. Chem. Soc. 1987, 109,7845-7854) or NH3/EtOH (Scaringe et al. Nucleic Acids Res. 1990, 18,5433-5341) for ˜20 h @ 55-65° C. Applicant has determined that the useof methylamine or NH₄OH/methylamine for 10-15 min @55-65° C. givesequivalent or better results. The following exemplifies the procedure.

Example 2 RNA and Ribozyme Deprotection of Exocyclic Amino ProtectingGroups Using Methylamine (MA) or NH₄OH/Methylamine (AMA)

[0074] The polymer-bound oligonucleotide, either trityl-on or off, wassuspended in a solution of methylamine (MA) or NH₄OH/methylamine (AMA)@55-65° C. for 5-15 min to remove the exocyclic amino protecting groups.The polymer-bound oligoribonucleotide was transferred from the synthesiscolumn to a 4 mL glass screw top vial. NH₄OH and aqueous methylaminewere pre-mixed in equal volumes. 4 mL of the resulting reagent was addedto the vial, equilibrated for 5 m at RT and then heated at 55 or 65° C.for 5-15 min. After cooling to −20° C., the supernatant was removed fromthe polymer support. The support was washed with 1.0 mL ofEtOH:MeCN:H₂O/3:1:1, vortexed and the supernatant was then added to thefirst supernatant. The combined supernatants, containing theoligoribonucleotide, were dried to a white powder. The same procedurewas followed for the aqueous methylamine reagent.

[0075] Table III is a summary of the results obtained using theimprovements outlined in this application for base deprotection.

[0076] The second step of the deprotection of RNA molecules may beaccomplished by removal of the 2′-hydroxyl alkylsilyl protecting groupusing TBAF for 8-24 h (Usman et al. J. Am. Chem. Soc. 1987, 109,7845-7854). Applicant has determined that the use of anhydrous TEA.HF inN-methylpyrrolidine (NMP) for 0.5-1.5 h @ 55-65° C. gives equivalent orbetter results. The following exemplifies this procedure.

Example 3 RNA and Ribozyme Deprotection of 2′-Hydroxyl AlkylsilylProtecting Groups Using Anhydrous TEA.HF

[0077] To remove the alkylsilyl protecting groups, theammonia-deprotected oligoribonucleotide was resuspended in 250 μL of 1.4M anhydrous HF solution (1.5 mL N-methylpyrrolidine, 750 μL TEA and 1.0mL TEA.3HF) and heated to 65° C. for 1.5 h. 9 mL of 50 mM TEAB was addedto quench the reaction. The resulting solution was loaded onto a Qiagen500® anion exchange cartridge (Qiagen Inc.) prewashed with 10 mL of 50mM TEAB. After washing the cartridge with 10 mL of 50 mM TEAB, the RNAwas eluted with 10 mL of 2 M TEAB and dried down to a white powder.

[0078] Table IV is a summary of the results obtained using theimprovements outlined in this application for alkylsilyl deprotection.

[0079] RNA and Ribozyme Purification

[0080] The method of this invention generally features HPLC purificationof ribozymes. An example of such purification is provided below in whicha synthetic ribozyme produced on a solid phase is blocked. This materialis then released from the solid phase by a treatment with methanolicammonia, subsequently treated with tetrabutylammonium fluoride, andpurified on reverse phase HPLC to remove partially blocked ribozyme from“failure” sequences (FIG. 9). Such “failure” sequences are RNA moleculeswhich have a nucleotide base sequence shorter to that of the desiredenzymatic RNA molecule by one or more of the desired bases in a randommanner, and possess free terminal 5′-hydroxyl group. This terminal5′-hydroxyl in a ribozyme with the correct sequence is still blocked bylipophilic dimethoxytrityl group. After such partially blocked enzymaticRNA is purified, it is deblocked by a standard procedure, and passedover the same or a similar HPLC reverse phase column to remove othercontaminating components, such as other RNA molecules or nucleotides orother molecules produced in the deblocking and synthetic procedures(FIG. 10). The resulting molecule is the native enzymatically activeribozyme in a highly purified form.

[0081] Below are provided examples of such a method. These examples canbe readily. scaled up to allow production and purification of gram oreven kilogram quantities of ribozymes.

Example 4 HPLC Purification, Reverse-Phase

[0082] In this example solid phase phosphoramidite chemistry wasemployed for synthesis of a ribozyme. Monomers used were2′-t-butyl-dimethylsilyl cyanoethylphosphoramidites of uridine,N-benzoyl-cytosine, N-phenoxyacetyl adenosine, and guanosine (GlenResearch, Sterling, Va.).

[0083] Solid phase synthesis was carried out on either an ABI 394 or380B DNA/RNA synthesizer using the standard protocol provided with eachmachine. The only exception was that the coupling step was increasedfrom 10 to 12 minutes. The phosphoramidite concentration was 0.1 M.Synthesis was done on a 1 μmol scale using a 1 μmol RNA reaction column(Glen Research). The average coupling efficiencies were between 97% and98% for the 394 model and between 97% and 99% for the 380B model, asdetermined by a calorimetric measurement of the released trityl cation.The final 5′-DMT group was not removed. After synthesis, the ribozymeswere cleaved from the CPG support, and the base and phosphotriestermoieties were deprotected in a sterile vial by incubation in dryethanolic ammonia (2 mL) at 55° C. for 16 hours.

[0084] The reaction mixture was cooled on dry ice. Later, the coldliquid was transferred into a sterile screw cap vial and lyophilized. Toremove the 2′-t-butyldimethylsilyl groups from the ribozyme the obtainedresidue was suspended in 1 M tetra-n-butylammonium fluoride in dry THF(TBAF), using a 20-fold excess of the reagent for every silyl group, for16 hours at ambient temperature. The reaction was quenched by adding anequal volume of a sterile 1 M triethylamine acetate, pH 6.5. The samplewas cooled and concentrated on a SpeedVac to half of the initial volume.

[0085] The ribozymes were purified in two steps by HPLC on a C4 300 Å 5μm DeltaPak column in an acetonitrile gradient.

[0086] The first step, or “trityl on” step, was a separation of5′-DMT-protected ribozyme(s) from failure sequences lacking a 5′-DMTgroup. Solvents used for this step were: A (0.1 M triethylammoniumacetate, pH 6.8) and B (acetonitrile). The elution profile was: 20% Bfor 10 minutes, followed by a linear gradient of 20% B to 50% B over 50minutes, 50% B for 10 minutes, a linear gradient of 50% B to 100% B over10 minutes, and a linear gradient of 100% B to 0% B over 10 minutes.

[0087] The second step was a purification of a completely deprotected,i.e. following the removal of the 5′-DMT group, ribozyme by a treatmentwith 2% trifluoroacetic acid or 80% acetic acid on a C4 300 Å 5 μmDeltaPak column in an acetonitrile gradient. Solvents used for thissecond step were: A (0.1 M Triethylammonium acetate, pH 6.8) and B (80%acetonitrile, 0.1 M triethylammonium acetate, pH 6.8). The elutionprofile was: 5% B for 5 minutes, a linear gradient of 5% B to 15% B over60 minutes, 15% B for 10 minutes, and a linear gradient of 15% B to 0% Bover 10 minutes.

[0088] The fraction containing ribozyme, which is in thetriethylammonium salt form, was cooled and lyophilized on a SpeedVac.Solid residue was dissolved in a minimal amount of ethanol and ribozymein sodium salt form was precipitated by addition of sodium perchloratein acetone. (K⁺ or Mg²⁺ salts can be produced in an equivalent manner.)The ribozyme was collected by centrifugation, washed three times withacetone, and lyophilized.

Example 5 HPLC Purification, Anion Exchange Column

[0089] For a small scale synthesis, the crude material was diluted to 5mL with diethylpyrocarbonate treated water. The sample was injected ontoeither a Pharmacia Mono Q® 16/10 or Dionex NucleoPac® column with 100%buffer A (10 mM NaClO₄). A gradient from 180-210 mM NaClO₄ at a rate of0.85 mM/void volume for a Pharmacia Mono Q® anion-exchange column or100-150 mM NaClO₄ at a rate of 1.7 mM/void volume for a DionexNucleoPac® anion-exchange column was used to elute the RNA. Fractionswere analyzed by a HP-1090 HPLC with a Dionex NucleoPac® column.Fractions containing full length product at ≧80% by peak area werepooled.

[0090] For a trityl-off large scale synthesis, the crude material wasdesalted by applying the solution that resulted from quenching of thedesilylation reaction to a 53 mL Pharmacia HiLoad 26/10 Q-Sepharose®Fast Flow column. The column was thoroughly washed with 10 mM sodiumperchlorate buffer. The oligonucleotide was eluted from the column with300 mM sodium perchlorate. The eluent was quantitated and an analyticalHPLC was run to determine the percent full length material in thesynthesis. The eluent was diluted four fold in sterile H₂O to lower thesalt concentration and applied to a Pharmacia Mono Q® 16/10 column. Agradient from 10-185 mM sodium perchlorate was run over 4 column volumesto elute shorter sequences, the full length product was then eluted in agradient from 185-214 mM sodium perchlorate in 30 column volumes. Thefractions of interest were analyzed on a HP-1090 HPLC with a DionexNucleoPac® column. Fractions containing over 85% full length materialwere pooled. The pool was applied to a Pharmacia RPC® column fordesalting.

[0091] For a trityl-on large scale synthesis, the crude material wasdesalted by applying the solution that resulted from quenching of thedesilylation reaction to a 53 mL Pharmacia HiLoad 26/10 Q-Sepharose®Fast Flow column. The column was thoroughly washed with 20 mMNH₄CO₃H/10% CH₃CN buffer. The oligonucleotide was eluted from the columnwith 1.5 M NH₄CO₃H/10% acetonitrile. The eluent was quantitated and ananalytical HPLC was run to determine the percent full length materialpresent in the synthesis. The oligonucleotide was then applied to aPharmacia Resource RPC column. A gradient from 20-55% B (20 mMNH₄CO₃H/25% CH₃CN, buffer A=20 mM NH₄CO₃H/10% CH₃CN) was run over 35column volumes. The fractions of interest were analyzed on a HP-1090HPLC with a Dionex NucleoPac® column. Fractions containing over 60% fulllength material were pooled. The pooled fractions were then submitted tomanual detritylation with 80% acetic acid, dried down immediately,resuspended in sterile H₂O, dried down and resuspended in H₂O again.This material was analyzed on a HP 1090-HPLC with a Dionex NucleoPac®column. The material was purified by anion exchange chromatography as inthe trityl-off scheme (vide supra).

Example 6 Ribozyme Activity Assay

[0092] Purified 5′-end labeled RNA substrates (15-25-mers) and purified5′-end labeled ribozymes (˜36-mers) were both heated to 95° C., quenchedon ice and equilibrated at 37° C., separately. Ribozyme stock solutionswere 1 μM, 200 nM, 40 nM or 8 nM and the final substrate RNAconcentrations were -1 nM. Total reaction volumes were 50 μL. The assaybuffer was 50 mM Tris-Cl, pH 7.5 and 10 mM MgCl₂. Reactions wereinitiated by mixing substrate and ribozyme solutions at t=0. Aliquots of5 μL were removed at time points of 1, 5, 15, 30, 60 and 120 m. Eachaliquot was quenched in formamide loading buffer and loaded onto a 15%denaturing polyacrylamide gel for analysis. Quantitative analyses wereperformed using a phosphorimager (Molecular Dynamics).

Example 7 One Pot Deprotection of RNA

[0093] Applicant has shown that aqueous methyl amine is an efficientreagent to deprotect bases in an RNA molecule. However, in a timeconsuming step (2-24 hrs), the RNA sample needs to be dried completelyprior to the deprotection of the sugar 2′-hydroxyl groups. Additionally,deprotection of RNA synthesized on a large scale (e.g., 100 μmol)becomes challenging since the volume of solid support used is quitelarge. In an attempt to minimize the time required for deprotection andto simplify the process of deprotection of RNA synthesized on a largescale, applicant describes a one pot deprotection protocol (FIG. 12).According to this protocol, anhydrous methylamine is used in place ofaqueous methyl amine. Base deprotection is carried out at 65° C. for 15min and the reaction is allowed to cool for 10 min. Deprotection of2′-hydroxyl groups is then carried out in the same container for 90 minin a TEA.3HF reagent. The reaction is quenched with 16 mM TEAB solution.

[0094] Referring to FIG. 13, hammerhead ribozyme targeted to site A issynthesized using RNA phosphoramadite chemistry and deprotected usingeither a two pot or a one pot protocol. Profiles of these ribozymes onan HPLC column are compared. The figure shows that RNAs deprotected byeither the one pot or the two pot protocols yield similar full-lengthproduct profiles. Applicant has shown that using a one pot deprotectionprotocol, time required for RNA deprotection can be reduced considerablywithout compromising the quality or the yield of full length RNA.

[0095] Referring to FIG. 14, hammerhead ribozymes targeted to site A(from FIG. 13) are tested for their ability to cleave RNA. As shown inthe FIG. 14, ribozymes that are deprotected using one pot protocol havecatalytic activity comparable to ribozymes that are deprotected using atwo pot protocol.

Example 8 Improved Protocol for the Synthesis of PhosphorothioateContaining RNA and Ribozymes Using 5-S-Alkyltetrazoles as ActivatingAgent

[0096] The two sulfurizing reagents that have been used to synthesizeribophosphorothioates are tetraethylthiuram disulfide (TETD; Vu andHirschbein, 1991 Tetrahedron Letter 31, 3005), and3H-1,2-benzodithiol-3-one 1,1-dioxide (Beaucage reagent; Vu andHirschbein, 1991 supra). TETD requires long sulfurization times (600seconds for DNA and 3600 seconds for RNA). It has recently been shownthat for sulfurization of DNA oligonucleotides, Beaucage reagent is moreefficient than TETD (Wyrzykiewicz and Ravikumar, 1994 Bioorganic Med.Chem. 4, 1519). Beaucage reagent has also been used to synthesizephosphorothioate oligonucleotides containing 2′-deoxy-2′-fluoromodifications wherein the wait time is 10 min (Kawasaki et al., 1992 J.Med. Chem).

[0097] The method of synthesis used follows the procedure for RNAsynthesis as described herein and makes use of common nucleic acidprotecting and coupling groups, such as dimethoxytrityl at the 5′-end,and phosphoramidites at the 3′-end. The sulfurization step for RNAdescribed in the literature is a 8 second delivery and 10 min wait steps(Beaucage and lyer, 1991 Tetrahedron 49, 6123). These conditionsproduced about 95% sulfurization as measured by HPLC analysis (Morvan etal., 1990 Tetrahedron Letter 31, 7149). This 5% contaminating oxidationcould arise from the presence of oxygen dissolved in solvents and/orslow release of traces of iodine adsorbed on the inner surface ofdelivery lines during previous synthesis.

[0098] A major improvement is the use of an activating agent,5-S-ethyltetrazole or 5-S-methyltetrazole at a concentration of 0.25 Mfor 5 min. Additionally, for those linkages which are phosporothioate,the iodine solution is replaced with a 0.05 M solution of3H-1,2-benzodithiole-3-one 1,1-dioxide (Beaucage reagent) inacetonitrile. The delivery time for the sulfurization step is reduced to5 seconds and the wait time is reduced to 300 seconds.

[0099] RNA synthesis is conducted on a 394 (ABI) synthesizer using amodified 2.5 μmol scale protocol with a reduced 5 min coupling step foralkylsilyl protected RNA and 2.5 min coupling step for 2′-O-methylatedRNA. A 6.5-fold excess (162.5 μL of 0.1 M=32.5 μmol) of phosphoramiditeand a 40-fold excess of S—ethyl tetrazole (400 μL of 0.25 M=100 μmol)relative to polymer-bound 5′-hydroxyl was used in each coupling cycle.Average coupling yields on the 394 synthesizer, determined bycalorimetric quantitation of the trityl fractions, was 97.5-99%. Otheroligonucleotide synthesis reagents for the 394 synthesizer:detritylation solution was 2% TCA in methylene chloride; capping wasperformed with 16% N-Methyl imidazole in THF and 10% aceticanhydride/10% 2,6-lutidine in THF; oxidation solution was 16.9 mM 12, 49mM pyridine, 9% water in THF. Fisher Synthesis Grade acetonitrile wasused directly from the reagent bottle. S-Ethyl tetrazole solution (0.25M in acetonitrile) was made up from the solid obtained from AppliedBiosystems. Sulfurizing reagent was obtained from Glen Research.

[0100] Average sulfurization efficiency (ASE) is determined using theformula:

ASE=(PS/Total)^(1/n−1)

[0101] where,

[0102] PS=integrated ³¹P NMR values of the P=S diester

[0103] Total =integration value of all peaks

[0104] n=length of oligo

[0105] Referring to tables V and VI, effects of varying the delivery andthe wait time for sulfurization with Beaucage's reagent is described.These data suggest that 5 second wait time and 300 second delivery timeis the condition under which ASE is maximum.

[0106] Using the above conditions a 36 mer hammerhead ribozyme issynthesized which is targeted to site A. The ribozyme is synthesized tocontain phosphorothioate linkages at four positions towards the 5′ end.RNA cleavage activity of this ribozyme is shown in FIG. 16. Activity ofthe phosphorothioate ribozyme is comparable to the activity of aribozyme lacking any phosphorothioate linkages.

Example 9 Protocol for the Synthesis of 2′-N-Phtalimido-NucleosidePhosphoramidite

[0107] The 2′-amino group of a 2′-deoxy-2′-amino nucleoside is normallyprotected with N-(9-flourenylmethoxycarbonyl) (Fmoc; Imazawa andEckstein, 1979 supra; Pieken et al., 1991 Science 253, 314). Thisprotecting group is not stable in CH₃CN solution or even in dry formduring prolonged storage at −20° C. These problems need to be overcomein order to achieve large scale synthesis of RNA.

[0108] Applicant describes the use of alternative protecting groups forthe 2′-amino group of 2′-deoxy-2′-amino nucleoside. Referring to FIG.17, phosphoramidite 17 was synthesized starting from2′-deoxy-2′-aminonucleoside (12) using transient protection withMarkevich reagent (Markiewicz J. Chem. Res. 1979, S, 24). Anintermediate 13 was obtained in 50% yield, however subsequentintroduction of N-phtaloyl (Pht) group by Nefken's method (Nefkens, 1960Nature 185, 306), desilylation (15), dimethoxytrytilation (16) andphosphitylation led to phosphoramidite 17. Since overall yield of thismulti-step procedure was low (20%) applicant investigated somealternative approaches, concentrating on selective introduction ofN-phtaloyl group without acylation of 5′ and 3′ hydroxyls.

[0109] When 2′-deoxy-2′-amino-nucleoside was reacted with 1.05equivalents of Nefkens reagent in DMF overnight with subsequenttreatment with Et3N (1 hour) only 10-15% of N and 5′(3′)-bis-phtaloylderivatives were formed with the major component being N-Pht-derivative15. The N,O-bis by-products could be selectively and quantitivelyconverted to N-Pht derivative 15 by treatment of crude reaction mixturewith cat. KCN/MeOH.

[0110] A convenient “one-pot” procedure for the synthesis of keyintermediate 16 involves selective N-phthaloylation with subsequentdimethoxytrytilation by DMTCI/Et3N and resulting in the preparation ofDMT derivative 16 in 85% overall yield as follows. Standardphosphytilation of 16 produced phosphoramidite 17 in 87% yield. One gramof 2′-amino nucleoside, for example 2′-amino uridine (US Biochemicals®part # 77140) was co-evaporated twice from dry dimethyl formamide (Dmf)and dried in vacuo overnight. 50 mis of Aldrich sure-seal Dmf was addedto the dry 2′-amino uridine via syringe and the mixture was stirred for10 minutes to produce a clear solution. 1.0 grams (1.05 eq.) ofN-carbethoxyphthalimide (Nefken's reagent, 98% Jannsen Chimica) wasadded and the solution was stirred overnight. Thin layer chromatography(TLC) showed 90% conversion to a faster moving products (10% ETOH inCHCl₃) and 57 μl of TEA (0.1 eq.) was added to effect closure of thephthalimide ring. After 1 hour an additional 855 μl (1.5 eq.) of TEA wasadded followed by the addition of 1.53 grams (1.1 eq.) of DMT-CI(Lancaster Synthesis®, 98%). The reaction mixture was left to stirovernight and quenched with ETOH after TLC showed greater than 90%desired product. Dmf was removed under vacuum and the mixture was washedwith sodium bicarbonate solution (5% aq., 500 mis) and extracted withethyl acetate (2×200 mis). A 25 mm×300 mm flash column (75 grams Merckflash silica) was used for purification. Compound eluted at 80 to 85%ethyl acetate in hexanes (yield: 80% purity: >95% by ¹HNMR).Phosphoramidites were then prepared using standard protocols describedabove.

[0111] With phosphoramidite 17 in hand applicant synthesized severalribozymes with 2′-deoxy-2′-amino modifications. Analysis of thesynthesis demonstrated coupling efficiency in 97-98% range. RNA cleavageactivity of ribozymes containing 2′-deoxy-2′-amino-U modifications at U4and/or U7 positions (see FIG. 1), wherein the 2′-amino positions wereeither protected with Fmoc or Pht, was identical. Additionally, completedeprotection of 2′-deoxy-2′-amino-Uridine was confirmed bybase-composition analysis. The coupling efficiency of phosphoramidite 17was not effected over prolonged storage (1-2 months) at lowtemperatures.

[0112] Other embodiments are within the following claims. TABLE ICharacteristics of Ribozymes Group I Introns Size: ˜200 to > 1000nucleotides. Requires a U in the target sequence immediately 5′ of thecleavage site. Binds 4-6 nucleotides at 5′ side of cleavage site. Over75 known members of this class. Found in Tetrahymena thermophila rRNA,fungal mitochondria, chioroplasts, phage T4, blue-green algae, andothers. RNAseP RNA (M1 RNA) Size: ˜290 to 400 nucleotides. RNA portionof a ribonucleoprotein enzyme. Cleaves tRNA precursors to form maturetRNA. Roughly 10 known members of this group all are bacterial inorigin. Hammerhead Ribozyme Size: ˜13 to 40 nucleotides. Requires thetarget sequence UH immediately 5′ of the cleavage site. Binds a variablenumber nucleotides on both sides of the cleavage site. 14 known membersof this class. Found in a number of plant pathogens (virusoids) that useRNA as the infectious agent (FIG. 1) Hairpin Ribozyme Size: ˜50nucleotides. Requires the target sequence GUC immediately 3′ of thecleavage site. Binds 4-6 nucleotides at 5′ side of the cleavage site anda variable number to the 3′ side of the cleavage site. Only 3 knownmember of this class. Found in three plant pathogen (satellite RNAs ofthe tobacco ringspot virus, arabis mosaic virus and chicory yellowmottle virus) which uses RNA as the infectious agent. Hepatitis DeltaVirus (HDV) Ribozyme Size: 50-60 nucleotides (at present). Cleavage oftarget RNAs recently demonstrated. Sequence requirements not fullydetermined. Binding sites and structural requirements not fullydetermined, although no sequences 5′ of cleavage site are required. Only1 known member of this class. Found in human HDV. Neurospora VS RNARibozyme Size: ˜144 nucleotides (at present) Cleavage of target RNAsrecently demonstrated. Sequence requirements not fully determined.Binding sites and structural requirements not fully determined. Only 1known member of this class. Found in Neurospora VS RNA.

[0113] TABLE II Large-Scale Synthesis Activator Amidite % Full[Added/Final] [Added/Final] Length Sequence (min) (min) Time* ProductA₉T T [0.50/0.33] [0.1/0.02] 15 m 85 A₉T S [0.25/0.17] [0.1/0.02] 15 m89 (GGU)₃GGT T [0.50/0.33] [0.1/0.02] 15 m 78 (GGU)₃GGT S [0.25/0.17][0.1/0.02] 15 m 81 C₉T T [0.50/0.33] [0.1/0.02] 15 m 90 C₉T S[0.25/0.17] [0.1/0.02] 15 m 97 U₉T T [0.50/0.33] [0.1/0.02] 15 m 80 U₉TS [0.25/0.17] [0.1/0.02] 15 m 85 A (36-mer) T [0.50/0.33] [0.1/0.02]15/15 m 21 A (36-mer) S [0.25/0.17] [0.1/0.02] 15/15 m 25 A (36-mer) S[0.50/0.24] [0.1/0.03] 15/15 m 25 A (36-mer) S [0.50/0.18] [0.1/0.05]15/15 m 38 A (36-mer) S [0.50/0.18] [0.1/0.05] 10/5 m 42

[0114] TABLE III Base Deprotection % Full Deprotection Time LengthSequence Reagent (min) T ° C. Product iBu(GGU)₄ NH₄OH/EtOH 16 h 55 62.5MA 10 m 65 62.7 AMA 10 m 65 74.8 MA 10 m 55 75.0 AMA 10 m 55 77.2iPrP(GGU)₄ NH₄OH/EtOH  4 h 65 44.8 MA 10 m 65 65.9 AMA 10 m 65 59.8 MA10 m 55 61.3 AMA 10 m 55 60.1 C₉U NH₄OH/EtOH  4 h 65 75.2 MA 10 m 6579.1 AMA 10 m 65 77.1 MA 10 m 55 79.8 AMA 10 m 55 75.5 A (36-mer)NH₄OH/EtOH  4 h 65 22.7 MA 10 m 65 28.9

[0115] TABLE IV 2′-O-Alkylsilyl Deprotection % Full Deprotection TimeLength Sequence Reagent (min) T ° C. Product A₉T TBAF 24 h 20 84.5 1.4 MHF 0.5 h 65 81.0 (GGU)₄ TBAF 24 h 20 60.9 1.4 M HF 0.5 h 65 67.8 C₁₀TBAF 24 h 20 86.2 1.4 M HF 0.5 h 65 86.1 U₁₀ TBAF 24 h 20 84.8 1.4 M HF0.5 h 65 84.5 B (36-mer) TBAF 24 h 20 25.2 1.4 M HF 1.5 h 65 30.6 A(36-mer) TBAF 24 h 20 29.7 1.4 M HF 1.5 h 65 30.4

[0116] TABLE V NMR Data for UC Dimers containing PhosphorothioateLinkage Synthesis # Type Delivery Eq. Wait ASE (%) 3524 ribo 2 × 3 s10.4 2 × 100 s  95.9 3525 ribo 2 × 3 s 10.4 2 × 75 s  92.6 3530 ribo 2 ×3 s 10.4 2 × 75 s  92.1 3526 ribo 1 × 5 s 08.6 1 × 300 s 100.0 3578 ribo1 × 5 s 08.6 1 × 250 s 100.0 3529 ribo 1 × 5 s 08.6 1 × 150 s  73.7

[0117] TABLE VI NMR Data for 15-mer RNA containing PhosphorothioateLinkages Synthesis # Type Delivery Eq. Wait ASE (%) 3581 ribo 1 × 5 s08.6 1 × 250 s  99.6 3663 ribo 2 × 4 s 13.8 2 × 300 s 100.0 3582 2′-O-Me1 × 5 s 08.6 1 × 250 s  99.7 3668 2′-O-Me 2 × 4 s 13.8 2 × 300 s  99.83682 2′-O-Me 1 × 5 s 08.6 1 × 300 s  99.8

[0118]

1 6 11 nucleic acid single linear The letter “N” stands for any base.“H” represents nucleotide C, A, or U. 1 NNNNUHNNNN N 11 28 nucleic acidsingle linear The letter “N” stands for any base. 2 NNNNNCUGANGAGNNNNNNC GAAANNNN 28 14 nucleic acid single linear The letter “N”stands for any base. The letter “Y” is U or C. The letter “H” is A, U,or C. 3 NNNYNGHYNN NNNN 14 50 nucleic acid single linear The letter “N”stands for any base. 4 NNNNNNCAUU ACANNNNNNN NNNNACAAAN NNNNNNNNNGAAGNNNNNNN 50 85 nucleic acid single linear 5 UGGCCGGCAU GGUCCCAGCCUCCUCGCUGG CGCCGGCUGG GCAACAUUCC 50 GAGGGGACCG UCCCCUCGGU AAUGGCGAAUGGGAC 85 176 nucleic acid single linear 6 GGGAAAGCUU GCGAAGGGCGUCGUCGCCCC GAGCGGUAGU AAGCAGGGAA 50 CUCACCUCCA AUUUCAGUAC UGAAAUUGUCGUAGCAGUUG ACUACUGUUA 100 UGUGAUUGGU AGAGGCUAAG UGACGGUAUU GGCGUAAGUCAGUAUUGCAG 150 CACAGCACAA GCCCGCUUGC GAGAAU 176

1. A process for purifying chemically synthesized RNA having one or morechemical modifications, comprising: (a) loading said RNA on to reversephase high-performance liquid chromatography (HPLC) column, wherein saidRNA comprises a 5′-protecting group; (b) eluting said RNA by passing asuitable buffer through said reverse phase column; (c) removing said5′-protecting group from said RNA; (d) loading said unprotected RNA ontoan anion exchange high-performance liquid chromatography (HPLC) column;(e) eluting said RNA by passing a suitable buffer through said anionexchange column; and (f) collecting the eluate from said anion exchangecolumn and recovering said RNA from said eluate, under conditions whichallow for the purification of said RNA.
 2. A process for purifyingchemically synthesized RNA having one or more chemical modifications,comprising: (a) loading said RNA onto an anion exchange high-performanceliquid chromatography (HPLC) column; (b) eluting said RNA by passing asuitable buffer through said column; and (c) collecting the eluate fromsaid column and recovering said RNA from said eluate, under conditionswhich allow for the purification of said RNA.
 3. A process for purifyingchemically synthesized RNA having one or more chemical modifications,comprising: (a) loading said RNA onto an anion exchange high-performanceliquid chromatography (HPLC) column; (b) eluting said RNA by passing asuitable buffer through said column; (c) collecting the eluate from saidcolumn and desalting said eluate; and (d) recovering said RNA from saiddesalted eluate under conditions which allow for the purification ofsaid RNA.
 4. A process for deprotecting and purifying chemicallysynthesized RNA having one or more chemical modifications, comprising:(a) contacting said RNA with an alkylamine under conditions suitable forremoving any exocyclic amine protecting groups or phosphate esterprotecting groups; (b) contacting said RNA with triethylamine-hydrogenfluoride under conditions suitable to remove any alkylsilyl protectinggroups from said RNA; (c) loading said RNA onto an anion exchangehigh-performance liquid chromatography (HPLC) column; (d) eluting saidRNA by passing a suitable buffer through said column; and (e) collectingthe eluate from said column and recovering said RNA from said eluate,under conditions which allow for the purification of said RNA.
 5. Aprocess for deprotecting and purifying chemically synthesized RNA havingone or more chemical modifications, comprising: (a) contacting said RNAwith an alkylamine under conditions suitable for removing any exocyclicamine protecting groups or phosphate ester protecting groups; (b)contacting said RNA with triethylamine-hydrogen fluoride underconditions suitable to remove any alkylsilyl protecting groups from saidRNA (c) loading said RNA onto an anion exchange high-performance liquidchromatography (HPLC) column; (d) eluting said RNA by passing a suitablebuffer through said column; (e) collecting the eluate from said columnand desalting said eluate; and (f) recovering said RNA from saiddesalted eluate under conditions which allow for the purification ofsaid RNA.
 6. The process of any of claim 1, wherein said anion exchangecolumn is selected from the group consisting of Pharmacia Mono Q® columnand Dionex NucleoPac® column.
 7. The process of any of claim 2, whereinsaid anion exchange column is selected from the group consisting ofPharmacia Mono Q® column and Dionex NucleoPac® column.
 8. The process ofany of claim 3, wherein said anion exchange column is selected from thegroup consisting of Pharmacia Mono Q® column and Dionex NucleoPac®column.
 9. The process of any of claim 4, wherein said anion exchangecolumn is selected from the group consisting of Pharmacia Mono Q® columnand Dionex NucleoPac® column.
 10. The process of any of claim 5, whereinsaid anion exchange column is selected from the group consisting ofPharmacia Mono Q® column and Dionex NucleoPac® column.
 11. The processof any of claim 1, wherein said anion exchange column comprises resinsthat are either quaternary or tertiary amino derivatized stationaryphases.
 12. The process of any of claim 2, wherein said anion exchangecolumn comprises resins that are either quaternary or tertiary aminoderivatized stationary phases.
 13. The process of any of claim 3,wherein said anion exchange column comprises resins that are eitherquaternary or tertiary amino derivatized stationary phases.
 14. Theprocess of any of claim 4, wherein said anion exchange column comprisesresins that are either quaternary or tertiary amino derivatizedstationary phases.
 15. The process of any of claim 5, wherein said anionexchange column comprises resins that are either quaternary or tertiaryamino derivatized stationary phases.
 16. The process of claim 11,wherein said resin is either silica-based or polystyrene based.
 17. Theprocess of claim 12, wherein said resin is either silica-based orpolystyrene based.
 18. The process of claim 13, wherein said resin iseither silica-based or polystyrene based.
 19. The process of claim 14,wherein said resin is either silica-based or polystyrene based.
 20. Theprocess of claim 15, wherein said resin is either silica-based orpolystyrene based.
 21. The process of any of claim 1, wherein said RNAis an enzymatic RNA.
 22. The process of any of claim 2, wherein said RNAis an enzymatic RNA.
 23. The process of any of claim 3, wherein said RNAis an enzymatic RNA.
 24. The process of any of claim 4, wherein said RNAis an enzymatic RNA.
 25. The process of any of claim 5, wherein said RNAis an enzymatic RNA.
 26. The process of claim 21, wherein said enzymaticRNA is in a hammerhead motif.
 27. The process of claim 22, wherein saidenzymatic RNA is in a hammerhead motif.
 28. The process of claim 23,wherein said enzymatic RNA is in a hammerhead motif.
 29. The process ofclaim 24, wherein said enzymatic RNA is in a hammerhead motif.
 30. Theprocess of claim 25, wherein said enzymatic RNA is in a hammerheadmotif.
 31. The process of any of claim 1, wherein said RNA comprises aplurality of chemical modifications.
 32. The process of any of claim 2,wherein said RNA comprises a plurality of chemical modifications. 33.The process of any of claim 3, wherein said RNA comprises a plurality ofchemical modifications.
 34. The process of any of claim 4, wherein saidRNA comprises a plurality of chemical modifications.
 35. The process ofany of claim 5, wherein said RNA comprises a plurality of chemicalmodifications.
 36. The process of any of claim 1, wherein said chemicalmodification is sugar modification.
 37. The process of any of claim 2,wherein said chemical modification is sugar modification.
 38. Theprocess of any of claim 3, wherein said chemical modification is sugarmodification.
 39. The process of any of claim 4, wherein said chemicalmodification is sugar modification.
 40. The process of any of claim 5,wherein said chemical modification is sugar modification.
 41. Theprocess of any of claim 1, wherein said chemical modification is basemodification.
 42. The process of any of claim 2, wherein said chemicalmodification is base modification.
 43. The process of any of claim 3,wherein said chemical modification is base modification.
 44. The processof any of claim 4, wherein said chemical modification is basemodification.
 45. The process of any of claim 5, wherein said chemicalmodification is base modification.
 46. The process of any of claim 1,wherein said chemical modification is phosphate backbone modification.47. The process of any of claim 2, wherein said chemical modification isphosphate backbone modification.
 48. The process of any of claim 3,wherein said chemical modification is phosphate backbone modification.49. The process of any of claim 4, wherein said chemical modification isphosphate backbone modification.
 50. The process of any of claim 5,wherein said chemical modification is phosphate backbone modification.51. The process of claim 36, wherein said sugar modification is2′-O-methyl modification.
 52. The process of claim 37, wherein saidsugar modification is 2′-O-methyl modification.
 53. The process of claim38, wherein said sugar modification is 2′-O-methyl modification.
 54. Theprocess of claim 39, wherein said sugar modification is 2′-O-methylmodification.
 55. The process of claim 40, wherein said sugarmodification is 2′-O-methyl modification.
 56. The process of claim 36,wherein said sugar modification is 2′-deoxy-2′-amino modification. 57.The process of claim 37, wherein said sugar modification is2′-deoxy-2′-amino modification.
 58. The process of claim 38, whereinsaid sugar modification is 2′-deoxy-2′-amino modification.
 59. Theprocess of claim 39, wherein said sugar modification is2′-deoxy-2′-amino modification.
 60. The process of claim 40, whereinsaid sugar modification is 2′-deoxy-2′-amino modification.
 61. Theprocess of claim 36, wherein said sugar modification is2′-deoxy-2′-fluoro modification.
 62. The process of claim 37, whereinsaid sugar modification is 2′-deoxy-2′-fluoro modification.
 63. Theprocess of claim 38, wherein said sugar modification is2′-deoxy-2′-fluoro modification.
 64. The process of claim 39, whereinsaid sugar modification is 2′-deoxy-2′-fluoro modification.
 65. Theprocess of claim 40, wherein said sugar modification is2′-deoxy-2′-fluoro modification.
 66. The process of claim 46, whereinsaid phosphate backbone modification is phosphorothioate modification.67. The process of claim 47, wherein said phosphate backbonemodification is phosphorothioate modification.
 68. The process of claim48, wherein said phosphate backbone modification is phosphorothioatemodification.
 69. The process of claim 49, wherein said phosphatebackbone modification is phosphorothioate modification.
 70. The processof claim 50, wherein said phosphate backbone modification isphosphorothioate modification.
 71. The process of any of claim 1,wherein said RNA is an antisense RNA.
 72. The process of any of claim 2,wherein said RNA is an antisense RNA.
 73. The process of any of claim 3,wherein said RNA is an antisense RNA.
 74. The process of any of claim 4,wherein said RNA is an antisense RNA.
 75. The process of any of claim 5,wherein said RNA is an antisense RNA.
 76. The process of any of claim 1,wherein said RNA is between 28 and 70 nucleotides long.
 77. The processof any of claim 2, wherein said RNA is between 28 and 70 nucleotideslong.
 78. The process of any of claim 3, wherein said RNA is between 28and 70 nucleotides long.
 79. The process of any of claim 4, wherein saidRNA is between 28 and 70 nucleotides long.
 80. The process of any ofclaim 5, wherein said RNA is between 28 and 70 nucleotides long.
 81. Theprocess of any of claims 76, wherein said RNA is between 30 and 40nucleotides long.
 82. The process of any of claims 77, wherein said RNAis between 30 and 40 nucleotides long.
 83. The process of any of claims78, wherein said RNA is between 30 and 40 nucleotides long.
 84. Theprocess of any of claims 79, wherein said RNA is between 30 and 40nucleotides long.
 85. The process of any of claims 80, wherein said RNAis between 30 and 40 nucleotides long.
 86. The process of any of claim1, wherein said RNA is chemically synthesized using solid phasesynthesis.
 87. The process of any of claim 2, wherein said RNA ischemically synthesized using solid phase synthesis.
 88. The process ofany of claim 3, wherein said RNA is chemically synthesized using solidphase synthesis.
 89. The process of any of claim 4, wherein said RNA ischemically synthesized using solid phase synthesis.
 90. The process ofany of claim 5, wherein said RNA is chemically synthesized using solidphase synthesis.
 91. The process of claim 86, wherein said solid phasesynthesis utilizes nucleoside monomers having a 5′-protecting group anda 3′-coupling group.
 92. The process of claim 87, wherein said solidphase synthesis utilizes nucleoside monomers having a 5′-protectinggroup and a 3′-coupling group.
 93. The process of claim 88, wherein saidsolid phase synthesis utilizes nucleoside monomers having a5′-protecting group and a 3′-coupling group.
 94. The process of claim89, wherein said solid phase synthesis utilizes nucleoside monomershaving a 5′-protecting group and a 3′-coupling group.
 95. The process ofclaim 90, wherein said solid phase synthesis utilizes nucleosidemonomers having a 5′-protecting group and a 3′-coupling group.
 96. Theprocess of claim 91, wherein said 5′-protecting group is dimethoxytritylgroup.
 97. The process of claim 92, wherein said 5′-protecting group isdimethoxytrityl group.
 98. The process of claim 93, wherein said5′-protecting group is dimethoxytrityl group.
 99. The process of claim94, wherein said 5′-protecting group is dimethoxytrityl group.
 100. Theprocess of claim 95, wherein said 5′-protecting group is dimethoxytritylgroup.
 101. The process of claim 91, wherein said 3′-coupling group isphosphoramidite group.
 102. The process of claim 92, wherein said3′-coupling group is phosphoramidite group.
 103. The process of claim93, wherein said 3′-coupling group is phosphoramidite group.
 104. Theprocess of claim 94, wherein said 3′-coupling group is phosphoramiditegroup.
 105. The process of claim 95, wherein said 3′-coupling group isphosphoramidite group.
 106. The process of claim 86, wherein said solidphase synthesis of RNA is carried out on controlled pore glass (CPG)solid support.
 107. The process of claim 87, wherein said solid phasesynthesis of RNA is carried out on controlled pore glass (CPG) solidsupport.
 108. The process of claim 88, wherein said solid phasesynthesis of RNA is carried out on controlled pore glass (CPG) solidsupport.
 109. The process of claim 89, wherein said solid phasesynthesis of RNA is carried out on controlled pore glass (CPG) solidsupport.
 110. The process of claim 90, wherein said solid phasesynthesis of RNA is carried out on controlled pore glass (CPG) solidsupport.
 111. The process of claim 86, wherein said solid phasesynthesis of RNA is carried out on polystyrene solid support.
 112. Theprocess of claim 87, wherein said solid phase synthesis of RNA iscarried out on polystyrene solid support.
 113. The process of claim 88,wherein said solid phase synthesis of RNA is carried out on polystyrenesolid support.
 114. The process of claim 89, wherein said solid phasesynthesis of RNA is carried out on polystyrene solid support.
 115. Theprocess of claim 90, wherein said solid phase synthesis of RNA iscarried out on polystyrene solid support.
 116. The process of claim 66,wherein said phosphorothioate modification is introduced using asulfurizing reagent.
 117. The process of claim 67, wherein saidphosphorothioate modification is introduced using a sulfurizing reagent.118. The process of claim 68, wherein said phosphorothioate modificationis introduced using a sulfurizing reagent.
 119. The process of claim 69,wherein said phosphorothioate modification is introduced using asulfurizing reagent.
 120. The process of claim 70, wherein saidphosphorothioate modification is introduced using a sulfurizing reagent.121. The process of claim 116, wherein said sulfurizing reagent isBeaucage reagent.
 122. The process of claim 117, wherein saidsulfurizing reagent is Beaucage reagent.
 123. The process of claim 118,wherein said sulfurizing reagent is Beaucage reagent.
 124. The processof claim 119, wherein said sulfurizing reagent is Beaucage reagent. 125.The process of claim 120, wherein said sulfurizing reagent is Beaucagereagent.
 126. The process of claim 4, wherein said alkylamine ismethylamine.
 127. The process of claim 5, wherein said alkylamine ismethylamine.